Severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV), an emerging infectious pathogen, caused a global outbreak in 2002-2003. Though it is under control, it may cause future epidemics and may be utilized by bioterrorists to attack innocent people. Therefore, it is essential to develop effective and safe vaccines for preventing future SARS outbreaks and for biodefense preparedness. Currently, most SARS vaccines under development consist of inactivated SARS-CoV or its full-length spike (S) protein. Concerns have been raised about the safety and efficacy of these vaccines since they may induce harmful immune responses or inflammatory reactions. We previously demonstrated that the receptor-binding domain (RBD), a small fragment in the S protein S1 domain responsible for SARS-CoV attachment, fusion and entry into the target cell contains multiple conformational epitopes that elicit highly potent neutralizing antibody responses and protective immunities against the SARS-CoV strains isolated from patients in 2002-2003 and 2003-2004 SARS epidemics and those from palm civets. Since RBD sequences in the S proteins of all SARS-CoV isolates identified so far exhibit more than 95% homology, we thus hypothesize that a recombinant RBD (rRBD) cloned and expressed in E. coli or yeast may elicit cross-neutralizing antibody responses and protective immunities against a majority of SARS-CoV strains that may cause future SARS outbreaks. Therefore, rRBD can be used as a subunit vaccine to protect people from SARS-CoV infection and prevent future SARS epidemics. The specific aims of this proposal are: (1) to express, purify and characterize rRBD of the SARS-CoV strains Tor2 and GD03T13 produced in E. coli or yeast. We will undertake a major effort in developing new strategies to enhance the yield and solubility of rRBD expressed in E. coli since E. coli remains the most preferable system for expressing large quantities of recombinant proteins for future preclinical and clinical studies. If it is proven that E. coli is not suitable for expression of rRBD, we will test yeast and other expression systems until an ideal expression system for production of rRBD is identified. Only the rRBD with proper antigenic conformation and biological activity will be selected for further study;(2) to assess the rRBD-induced antibodies against 3 representative SARS-CoV isolates;(3) to optimize the immunization regimens by selecting proper adjuvants, administration routes, and rRBD doses;and (4) to evaluate the in vivo efficacy and safety of the selected rRBD in mouse and ferret models. The long-term goal of this project is to develop highly effective and safe subunit vaccines for protecting at-risk populations from SARS-CoV infection or bioterrorism attack.